NOTE: End date changed to 03/31/2025 per NSSC information (Ed., 4/8/24).

The experiment (Advanced Plant Experiment 10) was launched on the NG-20 Cygnus spacecraft. All in orbit operations were successfully completed in the Veggie hardware on the International Space Station and all the biology samples were returned to Earth on the CREW 7 vehicle. [Ed. Note: The NASA Vegetable Production System (Veggie) is a plant growth unit on the International Space Station (ISS). It is capable of producing salad-type crops to provide the crew with a safe, palatable, and nutritious source of food, while also providing a tool to support recreation and relaxation.] The biological samples consisted of sterile Petri plates containing nutrient gel medium with either tomato seeds alone, tomato seeds inoculated with spores of T. harzianum, or Petri plates inoculated with fungal spores alone. A 2 mm pore size polyester mesh that was bounded by a 3 mm wide, 0.5 mm thick, 3d printed PETG perimeter rim was incorporated onto the top of the nutrient gel before planting of the seeds or inoculating with fungal spores to facilitate crew harvesting of plants and fungal colonies at the end of the experiment. Plants were grown under continuous light for 14 days in the Veggie hardware, with crew photography every other day. The experiment also included gas sampling of the air around the Veggies to allow stable carbon isotope analysis for calculation of photosynthetic and physiological parameters such as plant water use efficiency. On day 14, plates were opened in the Life Sciences Glovebox on the ISS, to ensure fungal containment, and biological materials were harvested, frozen between aluminum blocks conditioned to -160˚C, and stored in the Minus Eighty (Degrees Celsius) Laboratory Freezer for ISS (MELFI) for sample return.

On orbit data included imaging of the plants and fungi as they grew and these images reveal that plants showed shorter roots and enhanced lateral root production in spaceflight when compared to ground controls. Treatment with Trichoderma led to reduced primary root growth and increased root system branching with high growth rates of the Trichoderma. Cabin air was also sampled for comparison to an analysis of stable carbon isotope ratios of the biological materials and analysis indicates that the 13C:12C isotopic ratio is suppressed in spaceflight although carbon to nitrogen content is unchanged.

Biochemical analysis of the samples has included measurement of chlorophyll and carotenoids as indicators of photosynthetic pigment content, quantification of the stress pigment anthocyanin and measurement of the levels of malondialdehyde, a biochemical reporter of oxidative stress. These analyses have revealed that although spaceflight did not alter photosynthetic pigment accumulation, it did lead to a shoot-level signature of stress (significant anthocyanin and malondialdehyde (MDA) accumulation).

Further analysis for these samples includes transcriptomic analysis via RNAseq and glycomic profiling of the cell wall materials. Glycomics involves extracting cell walls and then using a panel of monoclonal antibodies against known wall epitopes to quantify wall polymer abundance using an Enzyme-Linked Immunosorbent Assay (ELISA)-based assay. Glycomic profiling has now been optimized for use with these spaceflight-related samples across multiple plant species.

CURRENT WORK:

Current work is focused on completing the full glycomic profile from the Advanced Plant Experiment 10 (APEX 10) spaceflight samples and a set of parallel clinostat grown tomato samples, along with ionomic profiling. Ionomics quantifies trace element composition of 15 minerals within the plant using Inductively Couple Plasma Mass Spectroscopy.

PRESENTATIONS AND OUTREACH:

These spaceflight-related projects continue to form a key part of the laboratory’s outreach efforts. They have been presented at venues ranging from the meeting of the International Space Life Science working Group in Liverpool, UK, to the American Society for Gravitational and Space Research, to the Plant Cell Dynamics meeting, to the Midwest sectional meeting of the American Society of Plant Biologists. This research has also formed the core of outreach to the general public at events including the University of Wisconsin’s Science Expeditions and the ‘Space Camp’ offered to 5th-8th graders at the Deke Slayton Space Museum in Sparta, Wisconsin. Interviews about the flight portion of this grant have been presented to media such as Wisconsin Public Radio, WORT radio, The Midwest Farm Report, Spectrum News and local new outlets and magazines such as the Wisconsin State Journal.

The experiment was successfully launched on January 30th, 2024 on the NG-20 Cygnus spacecraft. The biological samples consisted of sterile Petri plates containing nutrient gel medium with either tomato seeds alone, tomato seeds inoculated with spores of the beneficial, symbiotic fungus Trichoderma harzianum, or Petri plates inoculated with Trichoderma alone. A 2 mm pore size polyester mesh that was bounded by a thin 3 mm wide, 0.5 mm thick, 3d printed PETG perimeter rim was incorporated onto the top of the nutrient gel before planting of the seeds or spores to facilitate crew harvesting of plants and fungal colonies at the end of the experiment. These experiment samples were prepared at NASA Kennedy Space Center and then transported to the International Space Station (ISS) wrapped in foil. Transport was at 4°C to prevent premature seed or fungal spore germination. Upon reaching the ISS on February 5th 2024, the samples were unwrapped and installed in the growth chamber hardware consisting of two Veggies. [Ed. Note: Veggie" is the NASA Vegetable Production System.] Plants were grown under continuous light for 14 days, with photography by the crew every other day. These images showed that there was no seed germination or visible fungal growth from the Trichoderma at the point of installation in the Veggie. In contrast, two days later, the warming to cabin temperature and exposure to light led to seed germination, as evidenced by obvious visible protrusion of the embryonic root from the seeds. A small ~1 cm diameter hyphal mat was also evident radiating from either the site of inoculation on the Trichoderma only plates or from the seeds in the plates where the tomato seeds had been inoculated with Trichoderma spores. As the experiment progressed, the tomato seedlings exhibited continued root and shoot extension and developed to the stage of two expanded true leaves. An expansion of the fungal mycelium was evident, reaching the furthest extent of the Petri plate over approximately 8 days.

A parallel ground control was performed in the ISS environmental simulator chambers at Kennedy Space Center. Preliminary comparison of the images of the flight samples to these ground controls suggests both flight and ground experiments had similar time courses of plant germination and fungal growth. The root system of the flight samples showed a more random directionality than the downward gravitropic responses seen in the ground controls. The flight samples also appear to show increased root branching.

The experiment also included gas sampling of the air around the Veggies to allow CoInvestigator (CoI) David Hanson at the University of New Mexico to perform stable carbon isotope analysis. By combining data from stable carbon isotope analysis from the CO2 of the cabin air with isotope analysis from the plants grown in orbit, a calculation of parameters such as plant water use efficiency can be made. The cabin air was sampled at the installation in the Veggie and at the end of the experiment using Silonite coated gas sampling canisters. In addition, each Veggie had two Petri plates containing Zeolite installed. The Zeolite passively absorbs the cabin CO2, providing a second sampling strategy. At the end of the experiment, the Zeolite plates were placed in gas tight bags for sample return. Both the sampling canisters and the Zeolite plates performed as expected and were successfully stored at the end of the experiment to await sample return and ground-based isotope analysis.

On day 14 (the harvest day for the plants), the Petri plates containing the plants and fungi were transferred to the Life Science Glove Box to ensure containment of any fungal materials. The biological samples (plants and/or fungus) was harvested by peeling the polyester mesh from the gel surface. The plants and fungal hyphae remained adhered to the mesh, facilitating sample retrieval from the nutrient gel. Each mesh sample was then wrapped in foil and rapidly frozen by clamping between aluminum blocks conditioned to -160°C. All samples were successfully harvested, frozen, and were stored at -80°C in the Minus Eighty Life Science Freezer on the ISS (MELFI) prior to sample return.

All the biology samples were returned frozen, along with the gas samples, on the CREW 7 vehicle that splashed down on March 12th, 2024. All were successfully transferred to Kennedy Space Center and the gas samplers and Zeolite samples were shipped directly to the University of New Mexico for isotopic analysis. The plant and fungal samples were stored at -80°C at Kennedy Space Center prior to shipment to the University of Wisconsin-Madison for post-flight analyses. Ongoing post-flight analyses include extraction of nucleic acid to perform RNA sequencing (RNAseq) to catalog patterns of gene expression. Additionally, measurements of the levels of chlorophyll and the stress pigment anthocyanin are being performed alongside quantification of malondialdehyde, a biochemical measure of levels of oxidative stress in the plants. A subset of plant samples will also be sent to the University of New Mexico where stable carbon isotope analysis will be performed.

II. PROGRESS: Science and Experiment Verification Tests. The major focus for work in the reporting period has been to define protocols and analyses through a Science Verification Test (SVT) and Experiment Verification Test (EVT). The SVT was completed in June of 2022 and EVT in October 2022. Both tests were successful with a rating of excellent across all of the experiment’s success criteria.

Overview of procedures: The combination of SVT and EVT have allowed definition of experiment procedures for the flight experiment. Briefly, seeds, Trichoderma spores, or seeds inoculated with Trichoderma spores will be planted on 12 cm square Petri dishes containing gel growth medium supplemented with ½ strength Linsmaier and Skoog salts. A polyester mesh (1 mm mesh size) supported by a custom rim 3d printed in polyethylene tetraphthalate will be laid on the gel surface prior to planting. This aids in subsequent harvest of plants and fungal hyphae as the crew can lift off the mesh with all samples attached and intact for further processing. After planting, germination will be delayed for pre-flight operations by holding the plates at 4 °C. The samples will then be installed in the Veggie growth chamber and grown for 14 days with photography every other day.

At 14 days, the tomato seedling roots are close to the bottom of the plate, and this growth feature defines when harvest will occur. The plates are then opened and mesh with samples attached is removed, wrapped in foil, and snap frozen between aluminum bricks conditioned to -160 °C. These samples are then stored at -80 °C until analysis. Current work is aimed to modify the harvest procedures to allow them to operate inside the Life Science Glovebox. This approach is to provide an extra level of containment on orbit.

It is important to delay germination of both the tomato seeds and the Trichoderma spores before insertion into the Veggie on orbit. Testing has revealed that germination of both the tomato and Trichoderma is delayed by storage in the dark at 4 °C. This approach proves to be effective for 2 weeks, at which point the Trichoderma spores begin to germinate and grow.

Once installed in the Veggie at room temperature, the seeds and Trichoderma geminate and grow until the plants fill the 12 cm Petri plate growth space after 14 days. At this point, the plants are well developed with true leaves and highly branched root systems and the Trichoderma colonies have grown to cover ~ 50% of the growth area. Analysis of the images through the growth time course shows that inoculation with Trichoderma alters both growth of the primary root and branching patterns, as predicted from previous research on this interaction.

Biochemical Assays: In addition to growth analysis from the imaging described above, the frozen samples have been successfully assayed for chlorophyll and carotenoid content (as a measure of photosynthetic capacity), accumulation of anthocyanin (as a general stress marker) and levels of malondialdehyde, (as a biochemical marker for oxidative stress). Testing of all of these assays has been highly successful in both the SVT and EVT.

RNA quality and quantity: One major aim of this research is to perform transcriptional profiling of the samples using RNA-seq. To test how well the samples perform for these kinds of assays, quantitative polymerase chain reaction (qPCR) has been used as a proxy for full RNA-seq analyses. Frozen samples provided material for isolation of RNA of sufficient quantity and quality (average A260/280 of 2.00) to separate root and shoot samples (average of 3.6 µg RNA/root or shoot sample/plate) and analyze them for quantitative PCR. Three representative genes were assessed in SVT and EVT as proof-of-concept that the nucleic acid will be suitable for broad-scale molecular analyses: PRP1b is a pathogenesis-related protein that is known to be induced in response to successful Trichoderma infection of the plant roots, HSP22 is a heat shock protein that is a marker for oxidative stress responses, and NT is a nitrate transporter that should be unresponsive to Trichoderma infection. The reference gene for the qPCR analysis was Elongation Factor 1-alpha. These genes were successfully analyzed and preliminary analyses show significant elevation of PRP1b and HSP22 in plants treated with Trichoderma, whereas NT was unaffected.

Stable C-isotope analysis: In order to monitor photosynthetic parameters such as water use efficiency, stable isotope analysis is also planned to be used. To test the sampling method for these analyses, 4 gas samples were taken of the ambient atmosphere in the vicinity of the Veggie using Silonite MiniCans, with 2 samples at the start (day 0) of the experiment and 2 at the end (day 14). These samples were shipped to the University of New Mexico for analysis by the Hanson lab. All 4 samples were successfully analyzed, showing an average 12C:13C CO2 isotopic ratio of 25.6 +/- 4.6 per mil. These provide the baseline atmospheric measurements for use in modeling from tissue stable isotope content.

The major focus for work in the reporting period has been to optimize and define flight procedures and protocols. Extensive testing revealed that 12 cm square Petri dishes offered the required balance between plant and fungal growth. Harvesting of both tomato and fungal samples has also been optimized. The plants/fungus are grown on the surface of a nutrient gel. However, removing the plant and associated fungal hyphal mat intact was initially challenging, as the fungus adhered to the growth matrix. A series of surface substrates was therefore tested before settling on 1 mm pore size cotton fiber mesh, which allowed both normal plant growth and the lifting of the whole plant and fungal samples from the gel surface intact. The large pore size of this mesh likely provides enough area of direct contact between the plant roots and the Phytagel growth medium below the cloth to sustain "normal" plant growth. The mesh was laid on the nutrient gel (1/2 strength LS medium with 1% Phytagel) surface and then seeds were placed on this layer and the plants grown along the surface. This approach allowed the whole plant, with Trichoderma attached, to be harvested by peeling the cotton substrate off of the surface of the gel. These samples were then snap frozen by placing the entire sample in a foil bag and rapidly freezing using aluminum blocks conditioned to -160°C.

Delaying germination: Cold temperatures were optimized to delay germination of both the tomato seeds and the Trichoderma spores before insertion into the NASA Vegetable Production System (Veggie) in orbit. Germination of both the tomato and Trichoderma is delayed by storage in the dark at 4°C for 2 weeks. At this time point, the Trichoderma (but not the tomato seeds) begins to germinate and grow, setting the limit on the timing of storage prior to initializing the experiment in the Veggie.

Confirmation of fungal inoculant: Sequencing data from samples of the fungal isolates to be used from 2 diagnostic genes (ITS1/2 and TEF1) was BLASTed against both the National Institutes of Health (NIH) sequence databases and using the species identification tools at Trichokey.com used to confirm the fungal inoculant as T. harzianum.

RNA quality and quantity: Initial testing indicates a single 3-week-old tomato seedling from each plate will provide > 1µg of RNA of sufficient quality to analyze the required separate root and shoot samples by both RNA-seq and quantitative (polymerase chain reaction) PCR tests.

II. GROUND-BASED CHARACTERIZATION

In parallel with the efforts to define flight procedures, the interaction between T. harzianum and both tomato and Arabidopsis plants has been further characterized. A key observation is that, as proposed by several groups, the growth promoting factor of the interaction between plant and fungus seems to be delivered by volatiles from the fungus. Thus, increased growth and stress resilience in the plants have been evident when the Trichoderma is confined away from the plant using a spit plate system (i.e., plates with a divider separating fungal growth media from the plants). Unexpectedly, growth of the plant roots has been seen towards the fungus and fungus towards the plants, suggesting that the plant root may also be releasing volatiles that are detected by the fungus. Defining the potential mechanism of both of these directional growth responses is a key goal for the coming year as, at a practical level, it may be possible to grow the fungus remotely from direct contact with the plant and/or just deliver the fungal volatiles but still gain the beneficial effects on plant growth.

III. PRESENTATIONS AND OUTREACH

These spaceflight-related projects have been presented at multiple outreach events at venues -- ranging from colleges and universities such as Elizabethtown College, Oregon State University, and the Instituto de Biologia Experimental e Tecnologica in Oeiras, Lisbon, Portugal -- to outreach to the general public at events such as the University of Wisconsin’s Science Expeditions, the Friends of Allen Centennial Garden and Madison Master Gardeners, and the Sidmouth Science Festival in the UK, as well as to school students as part of "Space Camp" at the Deke Slayton Memorial Space and Bike Museum in Sparta, WI.

Specifically, we propose to:

(1) Analyze the kinetics of growth and the transcriptome, ionome, and photosynthetic responses of tomato plants to spaceflight.

(2) Compare these responses in plants growing with or without the beneficial root symbiotic microbe Trichoderma harzianum.

(3) Use comparisons to ground-based control experiments, clinostats, and random positioning machines to ask whether such changes are likely linked to the microgravity element of spaceflight and how far these ground-based microgravity analogs really do trigger responses seen in the spaceflight environment.